In nine strains of Clostridium innocuum, 3 beta-hydroxysteroid-dehydrogenating activities were detected. 3 beta, 7 alpha, 12 alpha-Trihydroxy- and 3 beta-hydroxy-12-keto-5 beta-cholanoic acids were identified as reduction products of the respective 3-keto bile acids by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. One strain was shown to contain a NAD-dependent 3 beta-hydroxysteroid dehydrogenase. Enzyme production was constitutive in the absence of added bile acids. The specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids, with trisubstituted acids being more effective than disubstituted ones. A pH optimum of 10.0 to 10.2 was found after partial purification by DEAE-cellulose chromatography. A molecular weight of about 56,000 was established. 3 beta-hydroxysteroid dehydrogenase activity was also found in the membrane fraction after solubilization with Triton X-100, suggesting that the enzyme was originally membrane bound. The enzyme reduced a 3-keto group in unconjugated and conjugated bile acids, lower Km values being demonstrated with disubstituted than with trisubstituted bile acids. Keto functions at C-7 and C-12 further reduced the Km value. The enzyme was found to be partially heat labile (86% inactivation at 50 degrees C for 10 min).